Abstract: :
Purpose: To examine changes in the retina of transgenic (TG)animals containing the mouse myocilin gene with the Tyr423Hispoint mutation. This mutation in the mouse gene correspondsto the Tyr437His mutation in the human myocilin gene which maybe the cause of severe juvenile open–angle glaucoma.Methods: This mutation was introduced into a BAC clone containingthe full length mouse myocilin gene. TG mice containing themutated BAC DNA were generated using a standard protocol. Wholemount retinas and paraffin sections were quantitatively analyzedafter immunostaining with antibodies against specific cell markers.Apoptosis was assessed using a TUNEL technique. Levels of Thy–1mRNA in the retina were estimated by in situ hybridization andreal–time PCR.Results: Since the loss of ganglion cells (GC) in the retinais a hallmark of glaucoma, we focused specifically on changesin the ganglion cell layer. In the central retina, no significantdifferences were observed between TG animals and their wild–type(WT) littermates at 6–8 months of age. However, degenerativechanges were consistently found in the ganglion cell layer inperipheral retinas of TG animals. These changes included theappearance of TUNEL–positive cells. TUNEL–positivecells were not detected in the WT retinas. Cell counts of hematoxylinand eosin stained sections of TG eyes showed a 5–10% decreasein the number of cells in the ganglion cell layer, comparedwith WT. A similar (5–15%) decrease in cell number wasobserved in eye sections and whole mount retinas stained withan antibody against the neuronal marker NeuN. However, the mostsignificant changes (up to 20% decrease) were observed in sectionsor whole mount retinas stained with antibodies against Brn3bor NF68, which specifically label GC. Real–time PCR analysisof mRNA levels of Thy–1, a specific marker to assess RGCdegeneration, showed a 2.0–2.9 fold decrease in the quantityof Thy1 mRNA in TG as compared with WT retinas. In situ hybridizationanalysis demonstrated selective reduction in Thy–1 mRNAexpression in RGC on the periphery of TG retina.Conclusions: Comparative characterization of the TG phenotypesuggests that the presence of the mouse myocilin gene with theTyr423His point mutation induces selective degeneration of RGCin peripheral retina. The degenerative changes in the retinaof TG animals resemble those observed in human glaucoma patients.